Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography

Abstract

Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.

Figure 1

Extended and contracted conformations of the T6SS sheath in M. xanthus visualized by correlated cryo-PALM-ECT. (a, d) Low-resolution EM images (grayscale background), cryo-PALM images (red and yellow foreground), slices from high-resolution 3-D cryotomograms (grayscale foreground), and segmentations of cellular structures (blue - tubular structures, green - filament bundles, white - spherical granules) superposed. The cryo-PALM images reveal VipA-PA-GFP localization (red: low; yellow: high precision), identifying the tubular structures as T6SSs. Scale bar in d 400 nm. (b, e) Tomographic slices through the tubular structures (blue) in a, d showing extended and contracted T6SS sheaths, respectively. (c, f) Cross-sectional views of b and e, respectively. Scale bar in f 50 nm (applies to b, c, e, f).

Figure 2

New T6SS structures identified by correlated cryo-PALM-ECT. (a) A very short loaded T6SS structure with baseplate attached to membrane. Superposed low-resolution EM image, cryo-PALM signal, high-resolution cryotomographic slice, and object segmentations as in Figure 1. (b) Tomographic slice through the tubular structure corresponding to the segmentation model shown in a. Features of the T6SS highlighted in blue (sheath) and cyan (baseplate). (c) Tomographic slice in b without highlights. (d) Cross-section view of the T6SS sheath in c. (e) A bent T6SS structure with additional sheet, distinguished from other tubular structures in the vicinity. Segmentation of the cryotomogram shows different tubular structures (individually colored blue, pink, and green) and spherical granules (white). Scale bar in e 400 nm (applies to a, e). (f, j, m) Segmented models of tubular structures shown in e. (g, k, n) Tomographic slices through the tubular structures corresponding to the segmentation models shown in f, j, and m, respectively. White arrows in g indicate a sheet adjacent to the hollow tube. (h, i) Tomographic slices of cross-sections h' and i' of the tubular structure shown in g. (l, o) Cross-sectional views of k and n, respectively. Diameters of the tubules shown in d, h, l, and o are 13, 14, 20, and 33 nm, respectively. Scale bar in o 50 nm (applies to b, c, d, g, h, i, k, l, n, o).